For many applications, mass spectra are acquired in linear time-of-flight mass spectrometers because of their particularly high detection sensitivity, even though the quality of the spectra from time-of-flight mass spectrometers with reflectors is actually incomparably superior. The reflector in the time-of-flight mass spectrometer compensates different initial ion velocities and therefore delivers a far better mass resolution and mass reproducibility.
The masses of the substances are calculated by the flight times of their ions, using a calibration curve. The calibration curve is determined before by using reference mixtures of known substances with known masses.
The inadequate quality of the mass spectra obtained by matrix-assisted laser desorption in linear time-of-flight mass spectrometers is principally due to the formation of ions which delivers ions of widely differing initial velocities. There is a broad distribution of the ion's initial velocities, resulting in different flight times of ions of the same kind, broadening the ion signal and thus deteriorating the mass resolution. And there is a scattering of the mean velocity of the ions, resulting in different mean flight times and thus in wrong mass values, after the flight times are converted to mass values using the calibration curve.
The spectrum quality with respect to mass resolution can be improved by the method of delayed acceleration of the ions, whereby ions with different initial velocities are time-focused at the location of the ion detector (A. Holle et al., U.S. Pat. No. 5,654,545 A). This time focusing at the ion detector strictly acts only for ions of a single mass in the mass spectrum; for all other ions the location of the time focusing is in front of or behind the detector. By taking special measures, this time focusing can be made to occur at the same location (the location of the ion detector) for ions of different masses, so that a mass spectrum is produced that delivers a uniform resolution over the entire spectrum (J. Franzen, DE 196 38 577 C1, U.S. Pat. No. 5,969,348 A), although the mass resolution in time-of-flight mass spectrometers operated in linear mode is only ever moderately good because of the release of energy as ions decompose, as described in more detail below.
Even if the delayed ion acceleration improves the resolution of the mass spectra, the method cannot fully eliminate the influence of the scattering mean initial ion velocities on the masses. The processes during ionization of the substances in the laser-induced vaporization cloud are not very easily reproducible; they depend greatly on the structural inhomogeneities of the microcrystalline sample after it has been prepared. The inhomogeneities force the operator to use slightly different laser energy density settings in the laser focus on the sample, and this variation in turn leads to different average initial velocities of the ions in the explosively expanding vaporization cloud. Furthermore, the uneven thickness of the sample preparation causes the formation of ions at differing initial potentials, with the result that they pass through different potential differences, and therefore absorb slightly different energies, according to the location where they were formed. These two effects both influence the flight times of the ions and cannot be corrected.
The acquisition of mass spectra with time-of-flight mass spectrometers generally requires a very large number of individual spectra. Each individual spectrum consists of a large sequence of digital values, each value being a digitized measurement of the ion current arriving at the detector. Measurements are usually made in equal time intervals. Modern mass spectrometers measure the ion current every half nanosecond, i.e. they measure in a rate of two gigahertz. The individual spectra are usually added together, measuring value by measuring value, to form a sum spectrum. The ions for each individual spectrum are generated by a laser shot. This procedure of generating sum spectra is made necessary by the low measuring dynamics in the individual spectrum. At least about 50, and in some cases even 1,000 or more, individual spectra are acquired; in general, a sum spectrum consists of several hundred individual spectra.
The different average initial velocities and total energies of the ions in the laser shots mean that conversion of the ions' flight times into mass values should really be carried out differently for each laser shot. A conversion algorithm always of the same type but with different parameter sets could be used. However, as the parameters for converting the individual spectra are not known, such a method can only be applied if some reference ion types whose exact masses are known occur in each mass spectrum.
In actual practice it is extremely seldom that this time-consuming individual conversion of each individual spectrum is used. Instead the operator trusts that at least the time-of-flight spectra of one sample preparation will match sufficiently so that the spectra can be added together, measurement by measurement. It is accepted that the mass resolution and signal-to-noise ratio will deteriorate. The reason for not carrying out this individual conversion is often to save time; but in many cases it is simply not possible, or it is inappropriate for analytical reasons, to add reference substances to the sample preparations for the purpose of individually recalibrating the individual spectra.
In the linear operating mode of a time-of-flight mass spectrometer, it is possible to detect not only the stable ions, but also the fragment ions from so-called “metastable” decompositions of the ions, and even neutral particles that are formed from the ion decompositions along the way. All these fragment ions and neutral particles which have resulted from a single parent ion species have the same velocity as the parent ions and therefore reach the ion detector at the same time. In many areas of application, this gives a ten-fold to hundred-fold detection sensitivity; this applies, for example, to the measurement of protein profiles when searching for biomarkers, or to the protein profiles of microorganisms for the purpose of their identification. For these applications, the energy of the desorbing and ionizing laser is raised, thereby increasing the ion yield, but also their instability. This increased detection sensitivity is of such decisive importance for many applications that many of the disadvantages of linear operation of time-of-flight mass spectrometers described above are accepted.
However, the disadvantages described mean that no cleanly comparable mass spectra are obtained. The mass spectra have distorted mass scales; ions of the same substance do not show the same mass value. It is difficult, for example, to create a good reference spectrum library for identifying microbes on the basis of their protein profiles. Spectra of the same microbes from different sample preparations do not match exactly, but display apparently different mass values for what are actually identical proteins. Deviations of up to one percent of the mass value have been observed.
If a good reference spectrum library is successfully created in spite of these difficulties, there are then problems for searching in the library because the acquired mass spectrum of a microbe can be randomly distorted along the mass scale, and therefore no mass spectrum with a sufficiently good match of the mass values and intensities is found in the library.
Apart from the disadvantages of distortion of the mass values, as described above, mass spectra of metastable ions acquired with linear time-of-flight mass spectrometers always have an inferior mass resolution. This is due to the decompositions of the ions. When an ion decomposes, a small excess of internal energy is always released as kinetic energy of the two ion fragments. Depending on the direction of the decomposition in relation to the direction of flight, the particles may be slightly accelerated or slightly decelerated, which results in smearing of the flight times of particles that have the same parent ion mass. This in turn reduces the mass resolution. This reduction in resolution is thus inseparably connected with the increase in detection sensitivity, and cannot, in principle, be removed. Mass resolutions in this case amount to only R=1,000 to R=2,000, compared to good reflector type mass spectra with R=20,000 to R=40,000.
Today, linear time-of-flight mass spectrometers have three principal applications:
in protein profile analysis when searching for “biomarkers” as indicators of certain stress situations of the body and for corresponding diagnostic procedures, such biomarkers are proteins which are up or down regulated by the stress;
in protein profile analysis for identifying microbes and
in mass spectrometric analysis of mutations of genetic material.
In all three applications, mass spectra up to high mass ranges of, for example, 20,000 Daltons are measured. Because of the low mass resolution, the isotope groups, which consist of ion signals that differ by one Dalton respectively, cannot be resolved in major parts of the mass spectrum. Therefore, only the envelopes of the isotope groups are measured, a fact that makes the mass determination and a corresponding calibration difficult. Furthermore, protein profile spectra in particular are very signal-intensive, with many overlapping ion signals, which greatly impedes the comparison of patterns. The protein profile spectra can certainly contain the ion signals of several hundred different proteins.
A method which allows the so-called “monoisotopic mass” to be calculated from an envelope of the isotope group of a protein has been elucidated in Patent DE 198 03 309 C1 (C. Köster, U.S. Pat. No. 6,188,064 B1).
Time-of-flight mass spectrometers with reflectors have a very much better mass resolving power, in particular because no fragment masses contribute to the mass spectrum and different ion energies are compensated for. Nevertheless, here too, distortions of the mass scale occur. Although mass resolutions far above R=20,000 can be achieved, the mass accuracy after the device has been well calibrated, but without recalibration of the individual mass spectrum, is only around 30 to 50 ppm. A recalibration of the individual mass spectrum using internal reference masses reaches a mass accuracy of 5 ppm and better. “Mass accuracy” is usually defined as standard deviation between the “true” and the measured mass values.
If no internal reference masses are available, the same problem occurs here as with linear time-of-flight mass spectrometers, but on a much finer scale.